Discovery of a novel ROCK2 ATP competitive inhibitor by DNA-encoded library selection.

Neurodegeneration disorders, such as Alzheimer's disease (AD), have garnered significant attention due to their impact on individuals and society as a whole. Understanding the mechanisms behind these disorders and developing effective therapy strategies is of utmost importance. One potential therapeutic target that has emerged is Rho-associated coiled-coil containing protein kinase 2 (ROCK2), as its accumulation and activity have been closely linked to memory loss. In this report, we present the findings of a recent discovery involving a new molecule that has the ability to competitively inhibit ROCK2 activity. This molecule was identified through the utilization of a DNA-encoded library (DEL) screening platform. Following selection against ROCK2, an off-DNA compound was synthesized and examined to ascertain its inhibitory properties, selectivity, mechanism of action, and binding mode analysis. From the screening, compound CH-2 has demonstrated an IC50 value of 28 nM against ROCK2, while exhibiting a 5-fold selectivity over ROCK1. Further analysis through molecular docking has provided insights into the specific binding modes of this compound. Our findings suggest that DEL selection offers a rapid method for identifying new inhibitors. Among these, the CH-2 compound shows promise as a potential ROCK2 inhibitor and warrants further investigation.

Discovery of N-(1,3,4-thiadiazol-2-yl)benzamide derivatives containing a 6,7-methoxyquinoline structure as novel EGFR/HER-2 dual-target inhibitors against cancer growth and angiogenesis.

Targeting EGFR and HER-2 is an essential direction for cancer treatment. Here, a series of N-(1,3,4-thiadiazol-2-yl)benzamide derivatives containing a 6,7-methoxyquinoline structure was designed and synthesized to serve as EGFR/HER-2 dual-target inhibitors. The kinase assays verified that target compounds could inhibit the kinase activity of EGFR and HER-2 selectively. The results of CCK-8 and 3D cell viability assays confirmed that target compounds had excellent anti-proliferation ability against breast cancer cells (MCF-7 and SK-BR-3) and lung cancer cells (A549 and H1975), particularly against SK-BR-3 cells, while the inhibitory effect on healthy breast cells (MCF-10A) and lung cells (Beas-2B) was weak. Among them, the hit compound YH-9 binded to EGFR and HER-2 stably in molecular dynamics studies. Further studies found thatYH-9could induce the release of cytochrome c and inhibit proliferation by promoting ROS expression in SK-BR-3 cells. Moreover,YH-9could diminish the secretion of VEGF and bFGF factors in SK-BR-3 cells, then inhibited tube formation and angiogenesis. Notably,YH-9could effectively inhibit breast cancer growth and angiogenesis with little toxicity in the SK-BR-3 cell xenograft model. Taken together,in vitroandin vivoresults revealed that YH-9 had high drug potential as a dual-target inhibitor of EGFR/HER-2 to inhibit breast cancer growth and angiogenesis.

Novel 4,5-dihydrospiro[benzo[c]azepine-1,1'-cyclohexan]-3(2H)-one derivatives as PARP-1 inhibitors: Design, synthesis and biological evaluation.

To further explore the research of novel PARP-1 inhibitors, we designed and synthesized a series of novel amide PARP-1 inhibitors based on our previous research. Most compounds displayed certain antitumor activities against four tumor cell lines (A549, HepG2, HCT-116, and MCF-7). Specifically, the candidate compound R8e possessed strong anti-proliferative potency toward A549 cells with the IC50 value of 2.01 µM. Compound R8e had low toxicity to lung cancer cell line. And the in vitro enzyme inhibitory activity of compound R8e was better than rucaparib. Molecular docking studies provided a rational binding model of compound R8e in complex with rucaparib. The following cell cycle and apoptosis assays revealed that compound R8e could arrest cell cycle in the S phase and induce cell apoptosis. Western blot analysis further showed that compound R8e could effectively inhibit the PAR's biosynthesis and was more effective than rucaparib. Overall, based on the biological activity evaluation, compound R8e could be a potential lead compound for further developing novel amide PARP-1 inhibitors.

Design, synthesis and biological evaluation of novel (E)-N-phenyl-4-(pyridine-acylhydrazone) benzamide derivatives as potential antitumor agents for the treatment of multiple myeloma (MM).

A series of novel (E)-N-phenyl-4-(pyridine-acylhydrazone) benzamide derivatives were designed, synthesized, and evaluated for their anti-proliferative activity against two different human cancer cell lines and one human normal cell line. Compound 8b had the best anti-proliferative activity (IC50 = 0.12 ± 0.09 µM, RPMI8226 cells) than the other compounds. And compound 8b had lower toxicity than imatinib. Flow cytometry analysis showed that compound 8b could arrest the cell cycle at the G0/G1 phase, and induce apoptosis of RPMI8226 cells by promoting mitochondrial ROS release, thereby effectively inhibiting cell proliferation. Our findings provided a promising lead compound 8b for further structural optimization and will be instructive for the discovery of more potent antitumor drugs with high selectivity and low toxicity.

The Multivalent Adhesion Molecule SSO1327 plays a key role in Shigella sonnei pathogenesis.

Shigella sonnei is a bacterial pathogen and causative agent of bacillary dysentery. It deploys a type III secretion system to inject effector proteins into host epithelial cells and macrophages, an essential step for tissue invasion and immune evasion. Although the arsenal of bacterial effectors and their cellular targets have been studied extensively, little is known about the prerequisites for deployment of type III secreted proteins during infection. Here, we describe a novel S. sonnei adhesin, SSO1327 which is a multivalent adhesion molecule (MAM) required for invasion of epithelial cells and macrophages and for infection in vivo. The S. sonnei MAM mediates intimate attachment to host cells, which is required for efficient translocation of type III effectors into host cells. SSO1327 is non-redundant to IcsA; its activity is independent of type III secretion. In contrast to the up-regulation of IcsA-dependent and independent attachment and invasion by deoxycholate in Shigella flexneri, deoxycholate negatively regulates IcsA and MAM in S. sonnei resulting in reduction in attachment and invasion and virulence attenuation in vivo. A strain deficient for SSO1327 is avirulent in vivo, but still elicits a host immune response.

Association of human papillomavirus type 58 with breast cancer in Shaanxi province of China.

A number of reports have identified HPV DNA in breast cancer specimens and HPV type 16, 18, 31, 33, 45, and 51 were more prevalent. HPV 58 was frequently detected in cervical cancer in Shaanxi China. The aim of the present study was to investigate whether HPV 58 present in breast cancer. 169 cases of breast cancer samples and 83 benign breast lesions were analyzed. Type specific primers and oligonucliotide probe were used for the detection of HPV 58 by conventional PCR and in situ hybridization techniques. The HPV 58 viral load were measured by qPCR. p16 protein expression were evaluated by immunohistochemistry. HPV 58 E7 DNA was detected in 25 out of 169 formalin fixed paraffin embedded breast cancer tissues (14.79%) by PCR, only 1 out of 83 non-malignant breast lesions showed positive (1.20%). The results of ISH showed that 17 out of 169 (10.06%) malignant samples were positive for HPV 58 E7, and only 1 out of 83 non-malignant lesions was positive. Positive p16 immunostaining was observed in all the HPV 58 E7 ISH positive cases, but 16 out of 98 cases with HPV negative were p16 positive. The presence of HPV 58 in both normal duct epithelial cells and carcinoma in situ along with its presence in the cancer cells of the same specimen indicated the possible causal role of HPV 58 in breast cancer. The findings provide a solid morphological evidence of the involvement of HPV 58 in breast cancer development.

The relationship of the tertiary lymphoid structures with the tumor-infiltrating lymphocytes and its prognostic value in gastric cancer.

Introduction: To explore the relationship between the tertiary lymphoid structures (TLSs) and tumor-infiltrating lymphocytes (TILs), and their distribution characteristics as well as the prognostic value in gastric cancer (GC). Material and methods: The TLSs and four subtypes of TILs were assessed by immunohistochemical (IHC) staining. The presence of MECA-79 positive high endothelial venules (HEVs) identified among the ectopic lymphocyte aggregation area in the GC tissue was defined as valid TLSs. The number of labeled TILs was observed in 5 fields of the most positive cells in the tumor center, invasive edge and within the TLSs, at a field of vision ×40. Results: The TLS distribution was significantly higher in the tumor invasive edge than the tumor center (p < 0.001). Similarly, the infiltrating density of CD8+ T cells and GrB+ T cells was statistically significantly higher in the tumor infiltrating edge than the tumor center. The total number of TILs and FOXP3+ T cells showed a contrary distribution. There was a positive correlation of the density of TLSs and TILs with both the location and the immune phenotype. A higher frequency of TILs and TLSs is often associated with favorable clinicopathologic parameters. Higher numbers of peri-TLSs (p = 0.007), peri-CD8+ (p = 0.019) and peri-GrB+TILs (p = 0.032) were significantly correlated with the favorable overall survival. Multivariate analysis revealed that the densities of TILs (p = 0.019) and TLSs (p = 0.037) were independent prognostic predictor for GC patients. Conclusions: We provide evidence that TLSs were positively associated with lymphocyte infiltration in GC. Thus, the formation of TLSs predicts advantageous immune system function and can be considered as a novel biomarker to stratify the overall survival risk of untreated GC patients.

A single-center, randomized, open-label, dose-escalation study to evaluate the pharmacokinetics of tacrine analogue octahydrogenacridine succinate tablets in healthy Chinese subjects.

The objectives of the present study were to investigate the pharmacokinetics (PK) of tacrine analogue octahydrogenacridine (OHA) succinate tablets in healthy Chinese subjects. A single-center, randomized, open-label, dose-escalation study was conducted in 42 healthy Chinese subjects. Part I of the study (n=30, 16 male and 14 female) evaluated the PK profiles of OHA in healthy Chinese subjects (aged 18-45 years) after single and multiple doses of 2 mg, 4 mg and 8 mg. Part II (n=10) assessed food effect on PK characteristics of OHA. The 10 participants of 4 mg dose group in part I were given another single dose of 4 mg OHA under fed condition. Part III (n=12, 7 male and 5 female) investigate PK behavior of OHA in elderly (aged 65-75 years) Chinese healthy subjects. In Part I, following a single- and multiple-dose, octahydrogenacridine succinate was rapidly absorbed with a median t(max) of 0.67 to 1.00 h, and was eliminated with a mean t1/2 of 2.27 to 2.98 h in all dose groups and did not appear to be dose dependent. In Part II, there were no significant differences in Cmax, AUC0-tn or tmax between 4 mg single dose fasting group and fed group (p=0.257, 0.153, 0.098, respectively). In Part III, both in single-dose and multiple-dose treatment, the main PK parameters of OHA in the elderly healthy Chinese subjects showed no statistical difference with those of non-elderly group. All these results indicated that OHA might be a promising drug under development for Alzheimer's disease (AD) therapy.

Analysis of immune status in gastric adenocarcinoma with different infiltrating patterns and origin sites.

Tumor infiltration pattern (INF) and tumor origin site were reported to significantly affect the prognosis of gastric cancer (GC), while the immune status under these contexts is not clear. In this study, we correlated the density and phenotype of tumor-infiltrating lymphocytes (TILs) with INF and the tumor origin site to reflect the biological behavior of tumors from a new perspective and also determined their effects on overall survival (OS) and other related clinicopathological features in archival samples of 147 gastric cancers with 10-year follow-up data. We found that the INFc growth pattern (an invasive growth without a distinct border) of GC lacked immune cell infiltration, particularly the cytotoxic T cells and their activated form. It is also significantly associated with an unfavorable prognosis (P < 0.001) and proximal site (P = 0.001), positive lymph node metastasis (P = 0.002), and later tumor-node-metastasis stage (P < 0.001). Moreover, the density and sub-type of TILs infiltration were significantly different in disparate differentiated areas for the tumor tissue with INFb. Compared with distal gastric cancer, proximal gastric cancers were prone to grow in an INFc pattern (P = 0.001) and infiltrated with fewer TILs, experiencing a shorter survival time (P = 0.013). Multivariate analysis showed that only the INF and the density of TILs were demonstrated to be the independent prognostic factors of OS for the GC. We concluded that GC with an aggressive growth pattern arising from proximal sites always had a weak immune response and resulted in a poor prognosis. The interaction between them and their synergistic or antagonistic effects in the development of tumors need to be further studied. This study opens up a new perspective for research on the biological behavior of the tumor.

Oncolytic Adenovirus H101 Synergizes with Radiation in Cervical Cancer Cells.

BACKGROUND: A major challenge in cervical cancer radiotherapy is tailoring the radiation doses efficiently to eliminate malignant cells and reduce the side effects in normal tissues. Oncolytic adenovirus drug H101 was recently tested and approved as a topical adjuvant treatment for several malignancies. OBJECTIVE: This study aimed to evaluate the potential neoadjuvant radiotherapy benefits of H101 by testing the inhibitory function of H101 in combination with radiation in different cervical cancer cells. METHODS: Human cervical cancer cell lines C33a, SiHa, CaSki, and HeLa were treated with varying concentrations of H101 alone or in combination with radiation (2 Gy or 4 Gy). Cell viability and apoptosis were measured at the indicated time intervals. HPV16 E6 and cellular p53 mRNA expression alteration was measured by qRT-PCR. In situ RNA scope was used to determine HPV E6 status. P53 protein alterations were detected by Western blot. RESULTS: Cell viability and apoptosis assays revealed that the combination of a high dose of H101 (MOI=1000, 10000) with radiation yielded a synergistic anticancer effect in all tested cervical cancer cell lines (P<0.05), with the greatest effect achieved in HPV-negative C33a cells (P<0.05). Low-HPV16-viral-load SiHa cells were more sensitive to the combination therapy than high-HPV16- viral-load CaSki cells (P<0.05). The combined treatment reduced HPV16 E6 expression and increased cellular P53 levels compared to those observed with radiation alone in SiHa and CaSki cells (P<0.05). CONCLUSION: Oncolytic adenovirus H101 effectively enhances the antitumor efficacy of radiation in cervical cancer cells and may serve as a novel combination therapy for cervical cancer.

Novel Allosteric Inhibitors of Deoxyhypusine Synthase against Malignant Melanoma: Design, Synthesis, and Biological Evaluation.

Based on the novel allosteric site of deoxyhypusine synthase (DHPS), two series of 30 novel 5-(2-methoxyphenoxy)-2-phenylpyrimidin-4-amine derivatives as DHPS inhibitors were designed and synthesized. Among them, compound 8m, with the best DHPS inhibitory potency (IC50 = 0.014 µM), exhibited excellent inhibition against melanoma cells, which was superior to that of GC7. Besides, molecular docking and molecular dynamics (MD) simulations further proved that compound 8m was tightly bound to the allosteric site of DHPS. Flow cytometric analysis and enzyme-linked immunosorbent assay (ELISA) showed that compound 8m could inhibit the intracellular reactive oxygen species (ROS) level. Furthermore, by western blot analysis, compound 8m effectively activated caspase 3 and decreased the expressions of GP-100, tyrosinase, eIF5A2, MMP2, and MMP9. Moreover, both Transwell analysis and wound healing analysis showed that compound 8m could inhibit the invasion and migration of melanoma cells. In the in vivo study, the tumor xenograft model showed that compound 8m effectively inhibited melanoma development with low toxicity.

Design, synthesis, and in vitro and in vivo anti-angiogenesis study of a novel vascular endothelial growth factor receptor-2 (VEGFR-2) inhibitor based on 1,2,3-triazole scaffold.

In the past five years, our team had been committed to click chemistry research, exploring the biological activity of 1,2,3-triazole by synthesizing different target inhibitors. In this study, a series of novel indole-2-one derivatives based on 1,2,3-triazole scaffolds were synthesized for the first time, and their inhibitory activity on vascular endothelial growth factor receptor-2 (VEGFR-2) was tested. Most of the compounds had shown promising activity in the VEGFR-2 kinase assay and had low toxicity to human umbilical vein endothelial cells (HUVECs). The compound 13d (IC50 = 26.38 nM) had better kinase activity inhibition ability than sunitinib (IC50 = 83.20 nM) and was less toxic to HUVECs. Moreover, it had an excellent inhibitory effect on HT-29 and MKN-45 cells. On the one hand, by tube formation assay, transwell, and Western blot analysis, compound 13d could inhibit VEGFR-2 protein phosphorylate on HUVECs, thereby inhibiting HUVECs migration and tube formation. In vivo study, the zebrafish model with VEGFR-2 labeling also verified that compound 13d had more anti-angiogenesis ability than sunitinib. On the other hand, molecular docking and molecular dynamics (MD) simulation results showed that compound 13d could stably bind to the active site of VEGFR-2. Based on the above findings, compound 13d could be considered an effective anti-angiogenesis drug and has more development value than sunitinib.

The special immune microenvironment of tumor budding and its impact on prognosis in gastric adenocarcinoma.

Recent studies showed that the tumor-infiltrating lymphocytes (TILs) are not randomly distributed, but organized to accumulate more or less densely in different regions within tumors, which have provoked new thoughts on cancer management. In this study we explored the characteristics of tumor immunemicroenvironment (TIME) for the tumor budding (TB) and lymphocytes in patients with gastric adenocarcinoma (GAC) as well as their prognostic significance. The TILs around the TB at the invasive margin were assessed by double-immunohistochemistry staining for the CD8, FOXP3, OX40 and GrB phenotypes. Results showed that there was a negative correlation between the density of TB and TILs in the budding area, tumor stroma and parenchyma. And the number of TILs around the TB was evidently reduced, compared with TILs in the non-budding region (P < 0.001). Additionally, the number of TILs in turn changed from non-budding area CD8+>FOXP3+>OX40+> GrB + T cells to FOXP3+>CD8+>OX40 + T > GrB + T cells in budding area. Survival rate was significantly lower in patients who had a higher density of TB (P < 0.001) and a lower density of TILs (P = 0.013). We concluded that TB was surrounded by a weak immune surveillance and immunosuppressive response supported the spatial heterogeneity in the TIME of gastric adenocarcinomas. The regional heterogeneity should be attached importance for identifying the influence of the TIME on cancer development and evolution.

Increased High-Risk Human Papillomavirus Viral Load Is Associated With Immunosuppressed Microenvironment and Predicts a Worse Long-Term Survival in Cervical Cancer Patients.

OBJECTIVES: To evaluate the correlation between tumor-infiltrating lymphocytes (TILs) and the viral load of high-risk human papillomavirus (HR-HPV) in cervical cancer patients. METHODS: A total of 62 cervical cancer patients were recruited during 1993-1994 and assigned into four groups treated with radiotherapy alone or radiotherapy combined with chemotherapy and/or thermotherapy. Ki67+ tumor cells, CD4+, CD8+, FoxP3+, OX40+ and granzyme B+ TILs were detected by immunohistochemistry. The viral load of HR-HPV in biopsy tissues before therapy was detected by in situ hybridization. RESULTS: The patients with high HPV viral load showed a significantly lower 15-year survival rate and an advanced International Federation of Gynecology and Obstetrics (FIGO) stage and increased recurrence rate. The distribution of Ki67+ tumor cells, FoxP3+ TILs, and CD8+/FoxP3+ ratio was obviously different between low and high HPV viral load groups. A worse clinical outcome was also implicated with increased HPV viral load tested by Cox regression analysis. CONCLUSIONS: Patients with increased HR-HPV viral load tend to be resistant to therapy with decreased immune surveillance in the immune microenvironment. Thus, HR-HPV viral load would influence the local immune microenvironment, and then further affect the survival of cervical cancer patients.

Development of a novel thymidylate synthase (TS) inhibitor capable of up-regulating P53 expression and inhibiting angiogenesis in NSCLC.

INTRODUCTION: The development of a new type of Thymidylate synthase (TS) inhibitor that could inhibit cancer cells' proliferation and anti-angiogenesis is of great significance for cancer's clinical treatment. OBJECTIVES: Our research hopes to develop a TS inhibitor that is more effective than the current first-line clinical treatment of pemetrexed (PTX) and provide a new reference for the clinical treatment of non-small cell lung cancer (NSCLC). METHODS: We obtained a series of novel TS inhibitors by chemical synthesis. Moreover, TS assay and molecular docking to verify the target compound's inhibitory mode. Use MTT assay, colony-forming assay, flow cytometry, and western blot to verify the compound's inhibitory effect on cancer cell proliferation and its mechanism; and explore the compound's effect on angiogenesis in vitro and in vivo. Further, explore the hit compound's anti-cancer ability through the xenograft tumor model and the orthotopic cancer murine model. RESULTS: A series of N-(3-(5-phenyl-1,3,4-oxadiazole-2-yl) phenyl)-2,4-dihydroxypyrimidine-5-sulfamide derivatives were synthesized as TS inhibitors for the first time. All target compounds significantly inhibited hTS enzyme activity and demonstrated significant antitumor activity against five cancer cell lines. Notably, 7f had a high selectivity index (SI) and unique inhibitory effects on eight NSCLC cells. In-depth research indicated that 7f could induce apoptosis by the mitochondrial pathway in A549 and PC-9 cells through the upregulation of wild-type P53 protein expression. Additionally, 7f was shown to inhibit angiogenesis in vitro and in vivo. In vivo studies, compared to PTX, 7f significantly inhibited tumor growth in A549 cell xenografts and had a higher therapeutic index (TGI). Moreover, 7f could prolong the survival of the orthotopic lung cancer murine model more effectively than PTX. CONCLUSION: The anti-angiogenic effect of 7f provides a new reference for the development of TS inhibitors and the clinical treatment of NSCLC.

Design, synthesis and biological evaluation of novel uracil derivatives bearing 1, 2, 3-triazole moiety as thymidylate synthase (TS) inhibitors and as potential antitumor drugs.

Research on thymidylate synthase inhibitors has been a hot spot for anticancer drug development. Here, based on the structures and pharmacological properties of two types of TS inhibitors, through a molecular assembly principle of drugs design, we designed and synthesized a series of 30 novel uracil derivatives as TS inhibitors. The antiproliferative ability of these compounds was evaluated against four cancer cell lines (A549, OVCAR-3, SGC-7901, and HepG2) by the MTT assay. Most of them showed excellent activities against all the tested cell lines. Furthermore, hTS assay results showed that these compounds have the unique ability to inhibit hTS activity in vitro. Notably, compound 13j exhibited the most potent activity against A549 cells (IC50 = 1.18 µM) and extremely prominent enzyme inhibition (IC50 = 0.13 µM), which was superior to the pemetrexed (PTX, IC50 = 3.29 µM and IC50 = 2.04 µM). Flow cytometric analysis showed the compound 13j could inhibit A549 cells proliferation by arresting the cell cycle in the G1/S phase, then induced the cell apoptosis. Further western blot analysis showed that compound 13j could down-regulate the cycle checkpoint proteins cyclin D1 and cyclin E to inhibit the cell cycle progression, and then induce intrinsic apoptosis by activating caspase-3, and reducing the ratio of bcl-2/bax. All of these results demonstrated that this new structure has potential drug-making properties and provides new ideas for drug development.

Discovery of N-phenyl-(2,4-dihydroxypyrimidine-5-sulfonamido) phenylurea-based thymidylate synthase (TS) inhibitor as a novel multi-effects antitumor drugs with minimal toxicity.

Thymidylate synthase (TS) is a hot target for tumor chemotherapy, and its inhibitors are an essential direction for anti-tumor drug research. To our knowledge, currently, there are no reported thymidylate synthase inhibitors that could inhibit cancer cell migration. Therefore, for optimal therapeutic purposes, combines our previous reports and findings, we hope to obtain a multi-effects inhibitor. This study according to the principle of flattening we designed and synthesized 18 of N-phenyl-(2,4-dihydroxypyrimidine-5-sulfonamido)phenyl urea derivatives as multi-effects inhibitors. The biological evaluation results showed that target compounds could significantly inhibit the hTS enzyme, BRaf kinase and EGFR kinase activity in vitro, and most of the compounds had excellent anti-cell viability for six cancer cell lines. Notably, the candidate compound L14e (IC50 = 0.67 µM) had the superior anti-cell viability and safety to A549 and H460 cells compared with pemetrexed. Further studies had shown that L14e could cause G1/S phase arrest then induce intrinsic apoptosis. Transwell, western blot, and tube formation results proved that L14e could inhibit the activation of the EGFR signaling pathway, then ultimately achieve the purpose of inhibiting cancer cell migration and angiogenesis in cancer tissues. Furthermore, in vivo pharmacology evaluations of L14e showed significant antitumor activity in A549 cells xenografts with minimal toxicity. All of these results demonstrated that the L14e has the potential for drug discovery as a multi-effects inhibitor and provides a new reference for clinical treatment of non-small cell lung cancer.

A case report of EGFR mutant lung adenocarcinoma that acquired resistance to EGFR-tyrosine kinase inhibitors with T790M mutation and epithelial-to-mesenchymal transition.

Although a secondary mutation and the epithelial-to-mesenchymal transition (EMT) are encountered very often in patients received the EGFR-TKI targeted treatment. The entire detrimental morphological change of the cancer entity was rare reported. Herein we report a case that acquired resistance to EGFR-TKI with T790M mutation and complete EMT morphological change of the tumor tissue. The primary lung tumor from a 52-year-old woman was diagnosed with moderate differentiated adenocarcinoma, with intensively positiveTTF-1 and E-cadherin in differentiated glandular structure but not the budding cancer cell cluster which with an intensive Vimentin staining. Molecular analysis revealed an EGFR exon 19 deletion and with an excellent response to Gefitinib treatment. Microscopic examination of recurred tumor specimens revealed a diffuse poorer differentiated proliferation of atypical cells. Immunostaining showed intensive Vimentin but almost completely negative for E-cadherin and TTF-1. Genetic analyses revealed T790M mutation. It is worth noting that rare clinical studies have been reported that acquired EGFR-TKI resistant lung adenocarcinoma underwent T790M mutation and almost complete EMT together. More significantly, the similarity of poorly differentiated cancer cell cluster in the primary lesions to recurred tumor lesions, which may pre-harbor drug resistance mutation should not be neglected underneath the predominant morphologic patterns.

Presence of high risk HPV DNA but indolent transcription of E6/E7 oncogenes in invasive ductal carcinoma of breast.

BACKGROUND AND AIMS: The causative role of high risk human papillomavirus (HR-HPV) in breast cancer development is controversial, though a number of reports have identified HR-HPV DNA in breast cancer specimens. Nevertheless, most studies to date have focused primarily on viral DNA rather than the viral transcription. The aim of this study was to investigate the presence of HR-HPV in breast cancer tissues at HPV DNA level and HPV oncogenes mRNA level by in situ hybridization (ISH). METHODS: One hundred and forty six (146) cases of breast invasive ductal carcinoma(IDC) and 83 cases of benign breast lesions were included in the study. Type specific oligonucleotide probes were used for the DNA detection of HPV 16,18 and 58 by ISH. HR-HPV oncogenes mRNA was assayed by novel RNAscope HR-HPV HR7 assay ISH. p16 protein expression was evaluated by immunohistochemistry (IHC). RESULTS: HR-HPV 16,18 and 58 DNA were detected in 52 out of 146 (35.6%) IDC and in 3 out of 83 (3.6%) benign breast lesions by ISH. The HR-HPV mRNAs was detected only in a few specimens with strong HPV DNA positivity(4/25) in a few scattered cancer cells with very weak punctate nuclear and/or cytoplasmic staining. p16 over-expression did not correlate with the HPV DNA positive breast cancer samples(17/52 HPVDNA+ vs 28/94 HPV DNA-, p=0.731). CONCLUSIONS: HR-HPVs certainly exist in breast cancer tissue with less active transcription, which implies that the causal role of HPV in breast cancer development need further study.

Surface display of the HPV L1 capsid protein by the autotransporter Shigella IcsA.

Autotransporters have become attractive tools for surface expression of foreign proteins in Gram-negative bacteria. In this study, the Shigella autotransporter IcsA, has been exploited to express the human papillomavirus (HPV) type 16 L1 capsid protein in Shigella sonnei and Escherichia coli. The L1 gene was fused in-frame to replace the coding sequence of the IcsA passenger domain that is responsible for actin-based motility. The resultant hybrid protein could be detected by an anti-L1 antibody on the surface of S. sonnei and E. coli. In E. coli, the protein was expressed on the entire surface of the bacterium. In contrast, the protein was detected mainly at one pole of the Shigella bacterium. However, the protein became evenly distributed on the surface of the Shigella bacterium when the icsP gene was removed. Our study demonstrated the possibility of exploiting autotransporters for surface expression of large, heterologous viral proteins, which may be a useful strategy for vaccine development.